In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially most important enzymes are proteases.
Although proteases have been used in the detergent industry for more than 30 years, much remains unknown as to details of how these enzymes interact with substrates and/or other substances present in e.g. detergent compositions. Some factors related to specific residues of the proteases and influencing certain properties, such as oxidative and thermal stability in general, of the proteases have been elucidated, but much remains to be found out. Also, it is still not exactly known which physical or chemical characteristics are responsible for a good washing performance or stability of a protease in a specific detergent composition.
The currently used proteases have for the most part been found by isolating proteases from nature and testing them in detergent formulations.
At present at least the following proteases are known to be commercially available and many of them are marketed in large quantities in many countries of the world.
Subtilisin BPN′ or Novo (available from e.g. SIGMA, St. Louis, U.S.A.), and Subtilisin Carlsberg, ALCALASE® (NOVO NORDISK A/S)) and MAXATASE®(Genencor).
A Bacillus lentus subtilisin, subtilisin 309, marketed by NOVO NORDISK A/S as SAVINASE®. A protein engineered variant of this enzyme is marketed as DURAZYM®.
Enzymes closely resembling SAVINASE®, such as subtilisin PB92, MAXACAL® marketed by Genencor Inc. (a protein engineered variant of this enzyme is marketed as MAXAPEM®), OPTICLEAN® marketed by SOLVAY et Cie. and PURAFECT® marketed by GENENCOR International.
A Bacillus lentus subtilisin, subtilisin 147, marketed by NOVO NORDISK A/S as ESPERASE®;
An increasing number of commercially used proteases are protein engineered variants of naturally occurring wild type proteases, e.g. DURAZYM® (Novo Nordisk A/S), RELASE® (Novo Nordisk A/S), MAXAPEM® (Gist-Brocades N.V.), PURAFECT® (Genencor International, Inc.).
Therefore, an object of the present invention, is to provide improved protein engineered protease variants, especially for use in the detergent industry.
Proteases
Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, San Francisco, Chapter 3). Bacteria of the Bacillus species secrete two extracellular types of protease, neutral proteases (or metalloproteases), and alkaline proteases among which the most important functionally is a serine endopeptidase and usually referred to as subtilisin.
Serine Proteases
A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 “Principles of Biochemistry,” Fifth Edition, McGraw-Hill Book Company, NY, pp. 271–272).
The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Daltons range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711–753).
Subtilases
A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al., Protein Engng. 4 (1991) 719–737. They are defined by homology analysis of more than 40 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilisins have been identified, and the amino acid sequence of a number of subtilisins have been determined. These include more than six subtilisins from Bacillus strains, namely, subtilisin 168, subtilisin BPN′, subtilisin Carlsberg, subtilisin Y, subtilisin amylosacchariticus, and mesentericopeptidase (Kurihara et al. (1972) J. Biol. Chem. 247 5629–5631; Wells et al. (1983) Nucleic Acids Res. 11 7911–7925; Stahl and Ferrari (1984) J. Bacteriol. 159 811–819, Jacobs et al. (1985) Nucl. Acids Res. 13 8913–8926; Nedkov et al. (1985) Biol. Chem. Hoppe-Seyler 366 421–430, Svendsen et al. (1986) FEBS Lett. 196 228–232), one subtilisin from an actinomycetales, thermitase from Thermoactinomyces vulgaris (Meloun et al. (1985) FEBS Lett. 198 195–200), and one fungal subtilisin, proteinase K from Tritirachium album (Jany and Mayer (1985) Biol. Chem. Hoppe-Seyler 366 584–492). for further reference Table I from Siezen et al. has been reproduced below.
Subtilisins are well-characterized physically and chemically. In addition to knowledge of the primary structure (amino acid sequence) of these enzymes, over 50 high resolution X-ray structures of subtilisins have been determined which delineate the binding of substrate, transition state, products, at least three different protease inhibitors, and define the structural consequences for natural variation (Kraut (1977) Ann. Rev. Biochem. 46 331–358).
One subgroup of the subtilases, I-S1, comprises the “classical” subtilisins, such as subtilisin 168, subtilisin BPN′, subtilisin Carlsberg (ALCALASE®, NOVO NORDISK A/S), and subtilisin DY.
A further subgroup of the subtilases I-S2, is recognized by Siezen et al. (supra). Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL®, Gist-Brocades NV), subtilisin 309 (SAVINASE®, NOVO NORDISK A/S), subtilisin 147 (ESPERASE®, NOVO NORDISK A/S), and alkaline elastase YaB.
Random and site-directed mutations of the subtilase gene have both arisen from knowledge of the physical and chemical properties of the enzyme and contributed information relating to subtilase's catalytic activity, substrate specificity, tertiary structure, etc. (Wells et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84; 1219–1223; Wells et al. (1986) Phil. Trans. R. Soc. Lond.A. 317 415–423; Hwang and Warshel (1987) Biochem. 26 2669–2673; Rao et al., (1987) Nature 328 551–554.
More recent publications covering this area are Carter et al. (1989) Proteins 6 240–248 relating to design of variants that cleave a specific target sequence in a substrate (positions 24 and 64); Graycar et al. (1992) Annals of the New York Academy of Sciences 672 71–79 discussing a number of previously published results; and Takagi (1993) Int. J. Biochem. 25 307–312 also reviewing previous results.
Especially site-directed mutagenesis of the subtilisin genes has attracted much attention, and various mutations are described in the following patent applications and patents:
Previously Characterized Protease Variants
Numerous references describe construction of Protease variants. In order to make it easier to get an overview of the overall prior art status, the references are ordered in two sections.
Section one deals with references describing the technological background and identification of Protease variants presently not believed to be related to the variants disclosed in the present invention. Those references are mainly included in order to summarize the state of the art within the field of construction of protease variants for different purposes.
Section two deals with references describing identification of Protease variants believed to be of some relevance to the variants of the present invention.
References Summarizing the State of the Art (Section 1):
EP 130756 (GENENTECH)(corresponding to U.S. Reissue Pat. No. 34,606 (GENENCOR)) relating to site specific or randomly generated mutations in “carbonyl hydrolases” and subsequent screening of the mutated enzymes for various properties, such as kcat/Km ratio, pH-activity profile, and oxidation stability. This publication claims site-specific mutation of subtilisins BPN′ in certain specified positions, i.e. −1Tyr, 32Asp, 155Asn, 104Tyr, 222Met, 166Gly, 64His, 169Gly, 189Phe, 33Ser, 221Ser, 217Tyr, 156Glu or 152Ala, to provide for enzymes exhibiting altered properties. Since these positions all except position −1 were known to be involved in the functioning of the enzyme prior to the filing of the application, this application does not contribute to solving the problem of deciding where to introduce mutations in order to obtain enzymes with specific desired properties.
EP 214435 (HENKEL) relating to cloning and expression of subtilisin Carlsberg and two mutants thereof. In this application no reason for mutation of 158Asp to 158Ser and 161Ser to 161Asp is provided.
In WO 87/04461 (AMGEN) it is proposed to reduce the number of Asn-Gly sequences present in the parent enzyme in order to obtain mutated enzymes exhibiting improved pH and heat stabilities, in the application emphasis is put on removing, mutating, or modifying the 109Asn and the 218Asn residues in subtilisin BPN′. No examples are provided for any deletions or for modifying the Gly-residues.
WO 87/05050 (GENEX) discloses random mutation and subsequent screening of a number of mutants of subtilisin BPN′ for improved properties. In the application mutations are described in positions 218Asn, 131Gly, 254Thr, 166Gly, 116Ala, 188Ser, 126Leu, and 53Ser.
EP 260105 (GENENCOR) describes modification of certain properties in enzymes containing a catalytic triad by selecting an amino acid residue within about 15 Å from the catalytic triad and replace the selected amino acid residue with another residue. Enzymes of the subtilase type described in the present specification are specifically mentioned as belonging to the class of enzymes containing a catalytic triad. In subtilisins positions 222 and 217 are indicated as preferred positions for replacement.
Also, it has been shown by Thomas, Russell, and Fersht (1985) Nature 318 375–376 that exchange of 99Asp into 99Ser in subtilisin BPN′ changes the pH dependency of the enzyme.
In a subsequent article (1987) J. Mol. Biol. 193 803–813, the same authors also discuss the substitution of 156Ser in place of 156Glu.
Both these mutations are within a distance of about 15 Å from the active 64His.
In Nature 328 496–500 (1987) Russel and Fersht discuss the results of their experiments and present rules for changing pH-activity profiles by mutating an enzyme to obtain changes in surface charge.
WO 88/08028 (Genex) and WO 88/08033 (Amgen) both relate to modifications of amino acid residues in the calcium binding sites of subtilisin BPN′. The enzyme is said to be stabilized by substituting more negatively charged residues for the original ones.
WO 95/27049 (SOLVAY S.A.) describes a subtilisin 309 type protease with the following mutations: N43R+N116R+N117R (BPN′ numbering. Data indicate the corresponding variant is having improved stability, compared to wildtype.
WO 95/30011, WO 95/30010, and WO 95/29979 (PROCTER & GAMBLE COMPANY) describe 6 regions, especially position 199–220 (BPN′ numbering), in both Subtilisin BPN′ and subtilisin 309, which are designed to change (i.e. decrease) the adsorption of the enzyme to surface-bound soils. It is suggested that decreased adsorption by an enzyme to a substrate results in better detergent cleaning performance. No data on any variants or specific detergent wash performance data are provided.
Previously Characterized Protease Variants Having Some Relevance to the Present Invention (Section Two):
In EP 251 446 (GENENCOR) it is described how homology considerations at both primary and tertiary structural levels may be applied to identify equivalent amino acid residues whether conserved or not. It is claimed that this information together with the inventors knowledge of the tertiary structure of subtilisin BPN′ brought the inventors to select a number of positions susceptible to mutation with an expectation of obtaining mutants with altered properties. The positions so identified are: 124Met, 222Met, 104Tyr, 152Ala, 156Glu, 166Gly, 169Gly, 189Phe, 217Tyr. Also 155Asn, 21Tyr, 22Thr, 24Ser, 32Asp, 33Ser, 36Asp, 46Gly, 48Ala, 49Ser, 50Met, 77Asn, 87Ser, 94Lys, 95Val, 96Leu, 107Ile, 110Gly, 170Lys, 171Tyr, 172Pro, 197Asp, 199Met, 204Ser, 213Lys, and 221Ser, which positions are identifed as being expected to influence various properties of the enzyme. Also, a number of mutations are exemplified to support these suggestions. In addition to single mutations in these positions the inventors also performed a number of multiple mutations. Further the inventors identify 215Gly, 67His, 126Leu, 135Leu, and amino acid residues within the segments 97–103, 126–129, 213–215, and 152–172 as having interest, but mutations in any of these positions are not exemplified.
Especially of interest for the purpose of the present invention the inventors of EP 251 446 suggest to substitute 170Lys (in subtilisin BPN′, type I-S1), specifically they suggest to introduce Glu or Arg for the original Lys. It appears that the Glu variant was produced and it was found that it was highly susceptible to autolytic degradation (cf. pages 48, 121, 123 (Table XXI includes an obvious error, but indicates a reduction in autolysis half-time from 86 to 13 minutes) and FIG. 32).
In WO 89/06279 (NOVO NORDISK A/S) position 170 is indicated as interesting and it is suggested to replace the existing residue with Tyr. However, no data are given in respect of such a variant. In WO 91/00345 (NOVO NORDISK A/S) the same suggestion is made, and it is shown that the Tyr variant of position 170 in subtilisin 309 (type I–S2) exhibits an improved wash performance in detergents at a pH of about 8 (variant S003 in Tables III, IV, V, VI, VIII, X). The same substitution in combination with other substitutions in other positions also indicates an improved wash performance (S004, S011–S014, S022–S024, S019, S020, S203, S225, S227 in the same Table and Table VII) all in accordance with the generic concept of said application.
In EP 525 610 A1 (SOLVAY) it is suggested to improve the stability of the enzyme (a type I-S2 subtilase closely related to subtilisin PB92) towards ionic tensides by decreasing the hydrophobicity in certain surface regions thereof. It is suggested to substitute Gin for the Arg in position 164 (170 if using BPN′ numbering). No variants comprising this substitution are disclosed in the application.
In WO 94/02618 (GIST-BROCADES N.V.) a number of position 164 (170 if using BPN′ numbering) variants of the I-S2 type subtilisin PB92 are described. Examples are provided showing substitution of Met, Val, Tyr, and Ile for the original Arg. Wash performance testing in powder detergents of the variants indicates a slight improvement. Especially for the lie variant wash performance tests on cacao an improvement of about 20–30% is indicated. No stability data are provided.
In PCT/DK96/00207 (not yet published) a number of protease variants with improved wash performance and/or storage stability is described.
It was found that subtilase variants with improved wash performance can by obtained by substituting one or more amino acid residues situated in, or in the vicinity of a hydrophobic domain of the parent subtilase for an amino acid residue more hydrophobic than the original residue, said hydrophobic domain comprising the residues corresponding to residues I165, Y167, Y171 of BLS309 (in BASBPN numbering), and said residues in the vicinity thereof comprising residues corresponding to the residues E136, G159, S164, R170, A194, and G195 of BLS309 (in BASBPN numbering) (PCT/DK96/00207).
U.S. Pat. No. 5,543,302 describes the identification of important autoproteolytic cleavage site in different wild type proteases (MILEZYME®, SAVINASE®, and ESPERASE®).
The present invention focuses on specific protease variants influencing the position of autoproteolytic cleavage sites in a group of protease variants described in our pending patent application PCT/DK96/00207. The variants of the present invention have surprisingly been found to exhibit an altered autoproteolytic degradation pattern compared to the autoproteolytic degradation pattern described for corresponding wildtype proteases in U.S. Pat. No. 5,543,302.